This invention relates to affinity ligands in general and, more particularly, to methods of producing highly selective matrices with reversible binding characteristics.
Separation and purification accounts for a very large percentage of the production cost of proteins and many specialty chemicals used for human therapeutics. Considerable effort is being expended on developing and optimizing techniques for the large-scale separation and purification of proteins. The difficulties lie in the high degrees of purity required for human therapeutics and therefore the extreme selectivity that is required for the separation process. These requirements result in complex, multi-step processes with concomitant high cost and low yield. Similar considerations hold true for specialty chemicals.
High selectivity in purification appears to be incompatible with low cost. For example, precipitation processes, or even ion-exchange chromatography, are relatively inexpensive operations, but they are also relatively nonselective and often must be accompanied by additional purification steps. Affinity separations often give a very high degree of purification in a single step, but biologically derived ligands such as monoclonal antibodies which are used in affinity chromatography are very costly, are unstable, and are not particularly easy to recycle or sterilize.
Affinity separations have been developed which exploit the affinity exhibited by proteins for metal ions. This property has been utilized in immobilized metal-affinity chromatography (IMAC) of proteins from natural sources and from recombinant organisms. A related technique, known as ligand-exchange chromatography, has been used in the purification of specialty chemicals such as amino acid derivatives and chiral precursors. In these techniques, a chelated metal ion with at least one available coordination site is covalently attached to a solid support and used as an affinity ligand to retain molecules which exhibit metal-coordinating moieties on their surfaces. Examples of such metal coordinating moieties are the amino acid side chains of histidine and cysteine on the surfaces of proteins. IMAC holds a number of important advantages over the use of biologically derived affinity ligands as recognition agents in protein separations. The small metal chelates generally used in metal-affinity separations are stable under a wide range of solvent conditions and temperatures. As a result, they can be recycled numerous times with negligible loss in performance. Other advantages of metal-affinity separations include the high metal loadings and therefore high protein capacities that can be attained and the relative ease of product elution and ligand regeneration. Proteins bound to chelated metals are easily removed by lowering the pH or by introducing a metal-binding ligand such as imidazole, and metal-affinity columns are regenerated simply by replenishing the supply of chelated metal. Metal chelate ligands have the additional advantage of being inexpensive.
Although metal-affinity separations are attractive from a number of economic and practical viewpoints, the metal-affinity ligands used are, unfortunately, not nearly as selective as biologically derived molecules such as antibodies. For example, chromatography on iminodiacetic acid-bound Cu(II), the most commonly used metal chelate, distinguishes proteins primarily by their surface histidine contents. While such current metal-affinity separations distinguish among proteins that contain widely different numbers of exposed histidines, it becomes more difficult to separate those with similar numbers of histidines.
One method to create polymeric matrices which exhibit selective binding interactions is to prepare polymers by a technique known as molecular imprinting or template polymerization. The technique is reviewed in Ekberg and Mosbach, TIBTECH 7:92-96 (1989), and in Wulff, Am. Chem. Soc. Symp. Ser. 308:186-230 (1986), and which describe molecular imprinting of small organic molecules and amino acids. Imprinting utilizes a template molecule with which to orchestrate the synthesis of individual monomers into a polymer matrix. The resulting matrix exhibits a large number of complementary interactions between the monomers and template molecule and can be viewed as a molecular "mold-like" structure. Such polymers are capable of specific recognition of template molecules and have been exclusively limited to small molecules.
A major disadvantage of this molecular imprinting technique is that the chemistry involved in synthesizing such polymer matrices is largely limited to organic solvents. While organic solvents can be used with small organic molecules and amino acids, they cannot be used with biological macromolecules or particles since they result in denaturation and inactivation of such molecules. Another disadvantage is that a large number of interactions are needed to selectively recognize a molecule as large as a protein. It is extremely difficult to synthesize materials with such a large number of complementary interactions.
There thus exists a need for inexpensive compositions which exhibit the high selectivity of biologically derived affinity ligands toward molecules, including macromolecules and biological particles, and yet retain reversible interactive and stability properties of metal-chelate ligands and imprinted polymers. The present invention satisfies these needs and provides related advantages as well.